Chromatography Forum

I would like to use a polyhydroxylethyl HILIC column (4.6 mm ID) as a step in a multi-dimensional natural peptide purification scheme.

I"ve been able to load VERY large volumes on RP columns (10 mL on a 1 mm column), and as long as the % organic in the sample is same or less than the starting mobile phase conditions, no loss of resolution occurred.

Can large sample volumes (1-2 mL) be loaded on a 4.6 mm HILIC column equilibrated with 85% MeCN + 10 mM NH4HOO pH 2.8 without loss of resolution?

As always the answer is it depends, theoretically peak compression should work better with HILIC than RP due to the steeper increase in retention as you decrease the percentage of strong eluent constituent (water in HILIC, acetonitrile in RP).
If your compound has decent retention at 85% AcN then you should be able to load (I have not tried loading more than 10% of the column volume) very large volumes in 95% AcN.

There is not a problem from the standpoint of the chromatography. The common issue in HILIC is a low solubility of the sample in the mobile phase. If this is not a problem for your analyte in the higher organic content, go for it. If it is a problem, consider at-column dilution, which we can discuss later if need arises.

I recently looked at some of these effects for simple basic pharmaceuticals on silica and cyano columns.

The problem was more complex than we expected. Buffer concentration was more important than acetonitrile for some analytes. Other analytes were not as sensitive to these effects. And equilibration times were very long on silica; much better on cyano. If your retention times are sensitive to buffer concentration, the problem gets worse.

I was looking at using "stronger" injection solvents, which is not the situation that you are considering, but remember if you have U-shaped retention patterns, the concept of "stronger" and "weaker" will depend on where you are on the U-shaped plot.

If you would like a copy of my Pittcon presentation email me privately.